中草药添加剂对禽流感病毒H9 N2感染肉鸡的保护试验

为了了解中草药添加剂“克侵灵”对禽流感(AIV)病毒H9N2感染肉鸡的保护效果,将1日龄AA鸡320羽随机分成不感染对照组、感染对照组、疫苗对照组、西药对照组、0.1%“克侵灵”组、0.2%“克侵灵”组、0.1%“克侵灵”+疫苗组、0.2%“克侵灵”+疫苗组。结果表明:“克侵灵”能阻止AIV H9N2的水平传播,提高鸡抵抗AIV H9N2攻击的能力,并能显著增加鸡的的体重。     

重组鸡α干扰素抑制H9N2禽流感病毒在鸡胚中增殖的研究

为检测重组鸡α干扰素(rChIFN-α)抑制H9N2禽流感病毒(AIV)在鸡胚中的增殖效力,本研究将rChIFN-α按其抗病毒效价高低分为4个组(10~7 U/mL、10~6 U/mL、10~5 U/mL和10~4 U/mL),同时设置阴性对照组和病毒对照组。各实验组再依次按照给药时间不同又分为"先给药再接种病毒组"、"同时给药和接种病毒组"及"先接种病毒再给药组"3个小组进行试验,通过检测H9N2 AIV血凝效价的变化、H9N2 AIV HA基因拷贝数以及各组EID_(50)评价rChIFN-α对H9N2 AIV增殖抑制效力。结果显示,除低剂量组以外,各剂量组与病毒对照组相比,结果均可以显著降低鸡胚尿囊液中H9N2 AIV的血凝效价(p0.01),同时能够显著减少H9N2 AIV基因的拷贝数(p0.01)及显著降低EID_(50)(p0.01)。表明中等剂量(10~5 U/mL)及更高剂量的rChIFN-α在鸡胚中能够显著抑制H9N2 AIV的增殖。本研究为rChIFN-α今后在临床中抗AIV治疗的实际应用提供了相关实验依据。     

中药"金不换"注射液在鸡胚中对鸡新城疫病毒的抑制试验

用鸡胚培养法和红血球凝集（HA）试验,测定中药“金不换”注射液对鸡新城疫病毒的抑制作用。试验共分4组,第Ⅰ组接种病毒尿囊液与中药金不换混合液,HA滴度0。第Ⅱ组接种病毒尿囊液与西药金刚烷胺混合液,HA滴度3log2。第Ⅲ组接种病毒尿囊液与生理盐水混合液,HA滴度9log2。第Ⅳ组不接种病毒尿囊液,不给药,HA滴度0。结果表明,中药“金不换”注射液对鸡胚无毒性作用,与病毒同时接种鸡胚,能完全抑制鸡新城病毒在鸡胚中增殖。     

2株H7N9亚型重组禽流感病毒的生物学特性和免疫原性研究

为评估经反向遗传构建的H7N9亚型重组禽流感病毒(AIV)H7-Re1株和H7-Re2株的生物学特性及免疫原性,本研究将这2株病毒分别以104倍稀释接种10日龄SPF鸡胚后,36℃培养,观察鸡胚存活情况并检测病毒HA效价,结果显示96 h时,2株病毒接种的鸡胚全部存活,培养至72 h时其HA效价均为9 log2,均显著高于亲本分离株。测定2株病毒的静脉内接种致病指数(IVPI),结果显示均为0。将2株病毒分别以10~6EID_(50)剂量鼻腔内接种5周龄SPF鸡,进行致病性试验,结果显示,接种后14 d内,2株病毒接种鸡均无临床症状,也不排毒。以2株病毒为种毒分别制备油乳剂灭活疫苗,以0.3 mL/只接种3周龄SPF鸡后3周时检测免疫鸡血清中HI抗体,结果显示,两种疫苗免疫鸡平均HI效价均可达到8.0 log2。于免疫后21 d,H7-Re1株免疫组鸡分别攻击10~6EID_(50)的H7N9亚型低致病力AIV(PG/SHH/S1069/13株)和10~5EID_(50)的高致病力AIV(CK/GX/SD098/17株),进行攻毒试验,结果显示两种病毒攻毒后免疫鸡均全部存活,且无发病和排毒出现。H7-Re2株免疫组鸡于免疫后21 d分别以10~5EID_(50)的剂量进行高致病力H7N9亚型AIV(CK/GX/SD098/17株)和H7N2亚型AIV(DK/FJ/SE0195/18株)的攻毒保护试验,结果显示两种病毒攻毒后免疫鸡均不发病、不死亡、不排毒。以上结果表明,以H1N1亚型A/Puerto Rico/8/34株病毒为内部基因供体、经反向遗传学方法构建的H7-Re1株和H7-Re2株均具有生长滴度高、生物安全性高和免疫原性好的特点。本研究为H7N9亚型禽流感疫苗的研制和应用奠定了基础。     

悬浮培养和鸡胚尿囊液抗原制备的H9亚型禽流感疫苗免疫效力比较试验

为了探讨悬浮培养和鸡胚尿囊液培养得到的H9亚型禽流感病毒(AIV)抗原制备的疫苗的免疫效力,试验用3株H9亚型AIV株分别接种微载体悬浮培养的MDCK细胞制备3种悬浮培养的细胞疫苗抗原,以3株H9亚型AIV株接种10日龄SPF鸡胚制备3种鸡胚尿囊液抗原,测定6种抗原液HA效价和鸡胚半数感染量(EID50);分别使用6种抗原液制备灭活疫苗,免疫21日龄SPF鸡,并在免疫后第21天采血测定HI抗体水平,采血后进行攻毒并在攻毒后第5天进行病毒分离试验。结果表明:两种方式制备的AIV抗原HA效价为7~9 lb,鸡胚半数感染量为1×106.9~7.9EID50,疫苗免疫后第21天用同源毒株作为工作抗原测定的免疫鸡HI效价差异不大,用异源毒株测定时有一定差异;两种方法得到的抗原制备的疫苗均能获得很好攻毒保护。     

中药蟾酥注射液鸡胚接种抗鸡新城疫病毒的试验研究

采用鸡胚培养法和血凝抑制试验,测定了2种中药配方对新城疫病毒的抑制作用。试验共5组,第1组接种病毒尿囊液与中药1号混合液,HA滴度0。第2组接种病毒尿囊液与中药2号混合液,HA滴度2 log2。第3组接种病毒尿囊液与金刚烷胺,HA滴度2 log2。第4组接种病毒尿囊液与生理盐水,HA滴度7.5log2。第5组不接种病毒尿囊液,不用药,HA滴度0。结果表明,中药1号配方好于2号配方,中药2号配方与金刚烷胺效果相同。     

中药组方鸡胚接种对鹅副粘病毒的抑制试验

选用中药组方对10日龄鸡胚进行药物毒性试验(第1组)和鹅副粘病毒抑制试验(第2组)。其中,第2组分为试验Ⅰ组(接种病毒尿囊液与中药组方混合液)、试验Ⅱ组(接种病毒尿囊液与西药利巴韦林混合液)、试验Ⅲ组(接种病毒尿囊液与生理盐水混合液)、对照组(不接种病毒尿囊液,不给药),并进行红血球凝集(HA)试验。结果:中药组方接种鸡胚无死亡;试验Ⅰ组HA滴度为0,试验Ⅱ组HA滴度为5 log2,试验Ⅲ组HA滴度为9 log2,对照组HA滴度为0。结果表明:中药组方对鸡胚无毒副作用;中药组方与病毒尿囊液同时接种鸡胚,能完全抑制鹅副粘病毒在鸡胚中增殖。     

共表达NDV F和IBDV VP0基因重组鸡痘病毒遗传稳定性研究

将共表达NDV F和IBDV VP0基因重组鸡痘病毒(重组鸡痘病毒vFV282)接种10日龄~11日龄SPF鸡胚,连续传10代,分别对第5、8、10代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282接种鸡胚成纤维细胞,分别对第1、5、10、15、20、25、30代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282翅翼刺种于鸡体上,连续传8代,对第3、5、8代传代毒进行病毒毒价测定和PCR鉴定。结果表明,重组鸡痘病毒vFV282在SPF鸡胚上传10代、在SPF鸡胚成纤维细胞传30代、在鸡体传8代后,其毒力未返强,F基因和VP0基因未发生缺失和变异。     

共表达NDV F和IBDV VPO基因重组鸡痘病毒遗传稳定性研究

将共表达NDV F和IBDV VP0基因重组鸡痘病毒(重组鸡痘病毒vFV282)接种10日龄～11日龄SPF鸡胚,连续传10代,分别对第5、8、10代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282接种鸡胚成纤维细胞,分别对第1、5、10、15、20、25、30代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282翅翼刺种于鸡体上,连续传8代,对第3、5、8代传代毒进行病毒毒价测定和PCR鉴定.结果表明,重组鸡痘病毒vFV282在SPF鸡胚上传10代、在SPF鸡胚成纤维细胞传30代、在鸡体传8代后,其毒力未返强,F基因和VP0基因未发生缺失和变异.     

“禽毒灵”研制与防治鸡传染性法氏囊病的应用

本试验研究证明，“禽毒灵”防治鸡传染性法氏囊病效果显著。跟踪追访户，用“禽毒灵”治疗病鸡15600只，治愈15325只，治愈率为96.85％－99.86％，平均为98.24%。对比试验结果表明，平均治愈率为“禽毒灵”试验组达97.00%，对照组为69.41%。试验组较对照组提高27.59%，卵黄抗体组较对照组提高26.59%。经统计学处理（X2检验），差异极显著（P＜0.001）。由此可见，“禽毒灵”略优于卵黄抗体。同时说明：“禽毒灵”还可防治鸡传染性支气管炎、喉气管炎、鸭病毒性肝炎等病毒性疾病。因此，“禽毒灵”具有开发利用和推广应用价值。     

Effect of a mannanoligosaccharide-supplemented diet on intestinal mucosa T lymphocyte populations in chickens challenged withSalmonella Enteritidis

Salmonellosis is one of the most common worldwide zoonosis, and the Enteritidis and Typhimurium serotypes are most prevalent. In this study, we evaluated the effects of a diet supplemented with mannanoligosaccharides (MOS) on CD3+, CD4+, CD8+, and goblet cell counts in the intestines of broilers vaccinated and challenged withSalmonella Enteritidis. The evaluation was based on randomized experiments including 45 chickens divided into 3 experimental groups. Group 1 received a nonsupplemented control diet. Group 2 received a diet supplemented with 1 kg MOS/t from d 1 to 21 and 0.5 kg MOS/t from d 22 to 56. Group 3 received a diet supplemented with 2 kg MOS/t from d 1 to 21 and 1 kg MOS/t from d 22 to 56. Chickens fed the lower level of MOS demonstrated reduced fecal shedding ofSalmonella 14 d after being challenged (P < 0.05). Both groups of MOS-supplemented chickens showed an increase in CD4+ and CD8+ cell counts in the ileal and cecal mucosa after being challenged withSalmonella Enteritidis (SE). Supplementation with MOS increased the specific T lymphocyte infiltration in the intestinal mucosa of chickens vaccinated and challenged withSalmonella Enteritidis.     

高效表达H9亚型禽流感病毒HA基因的重组鸡痘病毒的构建及免疫效力试验

将禽流感病毒血凝素 H9A基因克隆入插入载体 p FG11S中 ,通过酶切鉴定获得了正向转移载体 p FG11SHA;将其与禽痘病毒疫苗株 (w FPV)共转染鸡成纤维细胞 (CEF) ,通过蓝白斑筛选纯化得到重组病毒 r FPV- Ps- HA;以间接免疫荧光法证实 HA基因得到了表达。将该病毒经颈部皮下免疫 1日龄 SPF鸡 ,免疫后 15 d以 H9亚型禽流感病毒 F株翅静脉攻毒 ,攻毒后第 5天采集泄殖腔棉拭子样品进行病毒分离。将此重组病毒与以痘苗病毒 P7.5启动子表达相同基因的重组病毒 r FPV- P7.5 - HA作比较 ,结果表明 ,r FPV- Ps- HA相对于 r FPV- P7.5 - HA明显抑制了病毒的排出 ;攻毒后第 2、5、7、9、11天分别对 r FPV- Ps- HA、油乳剂灭活苗免疫鸡进行泄殖腔、气管排毒规律的检测 ,发现疫苗组均能很好地抑制排毒 ,攻毒对照组泄殖腔的排毒率明显高于气管排毒率     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.     

H5N1亚型禽流感病毒感染SPF雏鸡的外周血液免疫病理变化

以7日龄SPF雏鸡为试验动物,应用细胞培养和免疫酶技术,通过对外周血液免疫球蛋白含量、T和B淋巴细胞数量及其功能的检测,较全面系统地研究了鹅源H5N1亚型中强毒禽流感病毒(AIV)感染SPF雏鸡后,其外周血液上述指标的动态变化。结果发现,SPF雏鸡感染鹅源H5N1亚型AIV后,血清IgG和IgA含量在1~4d显著或极显著低于对照雏鸡(P<0.05或P<0.01),而IgM在病毒感染早期未见显著性差异,随后3种免疫球蛋白含量逐渐回升;血液T淋巴细胞数量显著低于对照雏鸡(P<0.05或P<0.01),而B淋巴细胞在1~5d明显降低(P<0.05或P<0.01);T、B淋巴细胞对ConA或PMA的增殖反应分别于1~8d、1~4d明显低于对照雏鸡(P<0.05或P<0.01)。上述结果表明,AIV感染SPF雏鸡外周血液无论是细胞免疫还是体液免疫功能均呈现一定抑制。     

Immunogenicity of an Escherichia coli multivalent pilus vaccine in chickens

Immunogenicity of an oil-emulsified Escherichia coli multivalent pilus vaccine was evaluated in 4-week-old chickens. The vaccine contained 180 micrograms of pilus protein from each of serotypes O1 and O78 and 170 micrograms of pilus protein from serotype O2. Chickens were vaccinated twice subcutaneously at 4 and 6 weeks old and challenged via the posterior thoracic air sac with E. coli serotype O1, O2, or O78 2 weeks after the last vaccination. Unvaccinated challenged chickens suffered 8% to 26% mortality; no vaccinated chickens died. Vaccinated chickens had very mild gross lesions in the air sacs, livers, and pericardial sacs and eliminated E. coli more efficiently than the unvaccinated challenged chickens. The results showed that a multivalent pilus vaccine protects chickens against active respiratory infection.     

Immunization of chickens and turkeys against avian influenza with monovalent and polyvalent oil emulsion vaccines.

Chickens and turkeys vaccinated with inactivated virus oil-emulsion vaccines containing different concentrations of either 1 (monovalent) or 4 (polyvalent) strains of avian influenza virus (AIV) were challenged-exposed with virulent AIV A/chicken/Scotland/59 or A/turkey/Ontario/7732/66. Four of 6 vaccines protected completely against postexposure mortality. Vaccine valency did not alter the serologic and challenge-exposure responses of chickens vaccinated with AIV A/turkey/Wisconsin/68, which was the virus component common to both monovalent and polyvalent vaccines. The magnitude of the serologic responses and protection against challenge-exposure were dependent on the concentration of virus in the vaccines. These data indicate that control of virulent AIV in chickens and turkeys by vaccination with inactivated vaccines may be feasible.     

Immunostimulant effect of a mixed herbal extract on infectious bursal disease virus (IBDV) vaccinated chickens in the context of a co-infection model of avian influenza virus H9N2 and IBDV

This study was conducted to assess the comparative effects of a mixed herbal extract (MHE) containing Ocimum sanctum, Withania somnifera, Emblica officinalis, Tinospora cordifolia, Mangifera indica, and Asphaltum (shilajit) on infectious bursal disease virus (IBDV)-vaccinated (VAC) chickens infected with IBDV and avian influenza virus (AIV) H9N2. The experiment included three groups (G1-G3): G1, the negative control group; G2, the VAC + challenged (Ch) group; and G3, the VAC + Ch + MHE group. MHE was orally administered continuously for 5 weeks post-vaccination (PV) with IBDV at 12 days of age, and the chicks were simultaneously challenged with virulent IBDV (intraocularly) and AIV H9N2 (intranasally) at 21 days PV. Blood and tissue samples as well as tracheal and cloacal swabs were gathered at different times PV and post-challenge. Immunological and haematological parameters, histopathological lesions, relative organ weights and final live weights revealed significant differences (P ≤ 0.05) between G2 and G3 groups. Furthermore, in the G3 group, the protection rates, ELISA and HI titers and CD4+/CD8+ ratio were significantly increased, whereas viral shedding titers and the heterophil/lymphocyte ratio were decreased. In conclusion, the oral administration of the mixed herbal extract for 5 weeks can stimulate the immune response to IBDV vaccination and relieves the pathogenicity of an AIV H9N2 and IBDV co-infection in chickens.     

Influence of inoculation dose, inoculation schedule, chicken age, and host genetics on disease susceptibility and development of resistance to Eimeria tenella infection

Various parasite- and host-related factors influencing disease susceptibility and development of protective immunity against Eimeria tenella infection were investigated in two inbred strains of chickens. Chickens that received a primary inoculation of 10(3), 10(4), or 10(5) oocysts showed a significant reduction in packed cell volume and produced significantly more oocysts than chickens inoculated with fewer oocysts. Younger chickens were as susceptible as older chickens to identical parasite doses. However, upon a secondary inoculation 5 weeks following primary inoculation, FP chickens 1 to 21 days old at the time of primary inoculation developed resistance to reinfection, whereas SC chickens less than 3 weeks old at the time of primary inoculation were highly susceptible to secondary infection. Flow cytometric analysis of spleen lymphocytes showed a substantial reduction in T-cell number in 1-day-old SC but not FP chickens. Furthermore, 1-week-old SC chickens showed depressed mitogenic responses to concanavalin A compared with 1-week-old FP chickens. There was no significant difference between SC and FP chickens in speen B-cell number, regardless of age.     

Salmonella flagellin enhances mucosal immunity of avian influenza vaccine in chickens

Flagellin, a bioactive Toll-like receptor (TLR) 5 ligand, may trigger the innate immunity that in turn is important for subsequent adaptive immune responses. In the present study, the adjuvant effects of the monomeric and polymeric forms of Salmonella flagellin (mFliC and pFliC, respectively) were examined in specific-pathogen free (SPF) chickens immunized intramuscularly (i.m.) or intranasally (i.n.) with formalin-inactivated avian influenza virus (AIV) H5N2 vaccines. Results showed that mFliC cooperating with the 64CpG adjuvant significantly induced influenza-specific antibody titers of plasma IgA in the i.m.-vaccinated animals. The nasal IgA levels in the i.n.-mFliC-coadministrated AIV vaccinated chickens were significantly elevated compared to levels observed in the control group (H5N2 vaccine alone). The pFliC cooperating with the 64CpG adjuvant significantly enhanced cell proliferation of splenocytes in the i.m.-vaccinated animals. TLR3 and TLR5 expressions were activated by flagellin stimulation in vitro and in vivo. These results suggest that flagellin can be used as an adjuvant in an AIV H5N2 vaccine, especially for mucosal immunity.